David Schaffer, Ph.D. Assistant Professor, Chemical Engineering and The Wills Neuroscience Institute University of California at Berkeley Berkeley, CA The Engineering and Evolution of Adeno-Associated Viral Gene Therapy Vectors for Improved Efficiency and Specificity Gene therapy, the introduction of genetic material to the cells of an individual for therapeutic benefit, has vast promise for translating our increasing knowledge of the molecular basis of disease into benefit for humanity. Gene therapy has achieved early clinical success recently and in one case has apparently cured several infants of a severe combined immunodeficiency disorder. However, a number of challenges must still be overcome before most diseases become accessible to gene therapy cures. In particular, the safety, efficiency, and stability of gene delivery vehicles require further improvement. Vectors based on adeno-associated virus (AAV) have been shown to be highly safe and efficient and have recently delivered therapeutic quantities of a transgene in a hemophilia clinical trial. Despite this success, AAV is not efficient for delivery to all cells, including several types of stem cells that are attractive targets for gene therapy. In particular, AAV is poor at delivery to neural stem cells, which are of particular interest to our research. However, we believe that the application of engineering principles will yield the necessary improvements in AAV performance. Therefore, guided by the disciplines of mass transfer, kinetics, and protein structure, we propose to analyze and identify barriers to AAV gene delivery to neural stem cells. Then, we will employ a combination of chemistry and molecular evolution approaches to overcome these barriers in order to enhance the efficiency and specificity of AAV gene delivery. In particular, the specific aims for this work are to: To determine heparin binding poses a barrier for AAV mass transfer through tissue To determine whether cell surface binding is a barrier for delivery to neural stem cells To explore whether endosomal escape is a limiting step for AAV delivery to neural stem cells To construct a mathematical model for computational analysis of the AAV gene delivery pathway We believe that the molecular engineering and evolution approach described in this work can help identify and overcome rate-limiting steps in the gene delivery pathway, from injection to expression, in order to enhance to performance of AAV vectors. Copyright 2001 The Whitaker Foundation 1700 North Moore Street, Suite 2200, Arlington VA 22209 (703) 528-2430     info@whitaker.org